Asymmetric porous adsorptive bead

ABSTRACT

The present invention relates to an asymmetric chromatography media suitable for separations applications, particularly as packed bed, fluidized bed or magnetized bed chromatography media. In certain embodiments, the asymmetric chromatography media comprises asymmetric particles, preferably beads, having at least two distinct, controlled pore size distributions. Preferably one of the distinct pore size distributions is in an internal region of the particle, and the other is in an external region or coating on the particle. These distinct pore size distributions can be modified with uniform or alternatively unique functional groups or mixtures of functional groups. The present invention allows for the control over pore size distribution within an asymmetric porous particle by providing a distinct internal region, preferably in the form of a bead, and a distinct external region, preferably in the form of a coating on the bead.

CROSS REFERENCE RELATED APPLICATIONS

The present utility patent application is a divisional patentapplication of U.S. patent application Ser. No. 11/520,848, filed onSep. 13, 2006, projected U.S. Pat. No. 7,678,269, projected Issue Dateof Mar. 16, 2010, which claims the benefit of priority of U.S.Provisional Patent Application No. 60/718,469, filed on Sep. 19, 2005,the entire content of each of which is incorporated herein in itsentirety.

BACKGROUND OF THE INVENTION

The present invention relates to asymmetric porous beads suitable forchromatographic use.

Biomolecules such as proteins, polypeptides and fragments ofbiomolecules have become important agents for pharmaceutical anddiagnostic applications. Currently, the purification of biomoleculesoften involves multiple steps, including chromatography. The synthesisand use of porous chromatography media for biomolecule separations iswell documented. Often the chromatographic steps are performed usingpacked beds of beads in which a product or impurity is separated fromthe feed stream. These beads are generally a base matrix (such as apolysaccharide, synthetic polymer or material, ceramic, glass or acomposite of the foregoing) that contains or has been modified withfunctionality that interacts with the biomolecule via chemical orphysical means; it provides a “driving force” for binding orinteraction. This complex set of interactions contributes to how themedia performs in the desired separation. The ability of the media toaffect a given separation between biomolecules is often referred to asthe “media's selectivity”. Typically these matrices exhibit porositythat allows for the biomolecule of interest to access the internalvolume of the particle. This internal porosity is uniform through theparticle.

Chromatographic separations typically are carried out in columns packedwith the separation matrix in form of particulate beads. The size of themedia particles dictates the kinetics of the separation, but smallerparticles can result in high back pressure. To be able to separate largemolecules the particles should have large pores, but large pores reducethe mechanical stability of the particles, particularly withpolysaccharides such as agarose. Polysaccharides are advantageousbecause they are typically low protein binding, easy to functionalize,and can form porous structures. Conventionally, polysaccharide beads aretypically made from one polysaccharide with a uniform or “symmetric”pore structure throughout the bead. Examples of these “symmetric” beadsinclude Cellufine® (cellulose), Sepharose® (agarose) and Sephadex®(dextran). These symmetric beads bind molecules throughout the internalstructure of the bead. The chemical environment (pore size,hydrophobicity, ligand type and ligand density) are essentially uniformwithin the internal structure. Therefore, the nature of the bindingenvironment throughout the bead is uniform. In these systems, thedriving force for separation comes from the difference in bindingstrength between absorbed biomolecules. Typically when a symmetric resinis used, optimization of the binding strength through control of thebuffer conditions is the only driving force for achieving the desiredseparation. In addition, polysaccharide materials are inherentlycompressible, and often require chemical modification to reducecompressibility such as through crosslinking.

Another property of a symmetric resin is the mechanical strength of themedia, which is a result of the material, pore structure and chemicalmodification. Typically for polysaccharides, the smaller the pore sizeof the media, the greater the mechanical strength due to higherconcentration of solid material (lower porosity). However, the smallerthe pore size, the more hindered the mass transfer of larger species(such as IgG) in to the adsorbent. Therefore, symmetric media designoften involves the optimization/trade-off of particle rigidity andbiomolecule mass transfer. This optimization problem is furthercomplicated by the fact that the permeability of a symmetric bead is theresult of the size and size distribution of the particle. As theparticle size gets smaller, external particle surface area increases andtherefore so does mass transfer. During elution, the smaller particleallows for a shorter diffusion time out of the media, thus causing amore concentrated, narrow elution peak. However, this improvement of themass transfer is at the expense of permeability, which restricts columndimensions and media throughput.

In many biomolecule separations, the species of interest (targetmolecule) is the most concentrated species in the feedstock. In otherwords, the impurities are only a small percentage of the total mixtureto be separated. With symmetric chromatography media, the separation isoften affected by binding the target molecule and some of theimpurities, and then separating the impurity by using the elutioncondition to differentiate the species. If the two species are verysimilar in their binding strength to the media, even if the proteins aredifferent in size, the separation can be very difficult. In some cases,the difficult nature of the separation is the result of the limitednumber of driving forces affecting the purification. The end result iseither a lower yield of purified protein from incomplete separation orthat another chromatography step is then essential to further purify thetarget molecule using a different media with different driving forcesfor separation. This is time-consuming, inefficient and expensive.

Filled polysaccharide beads are well established for uses includingexpanded bed or fluidized bead chromatography. Typically, thesematerials are made by adding a solid or non-porous sphere to thepolysaccharide (typically agarose) during bead formation. In thismanner, a bead with one or more non-porous particles encapsulated insidethe polysaccharide material can be formed. Another common technique isto coat the solid particle with a material that eventually becomes theabsorbent. The solid particle serves no function in the protein capacityor separation properties of the bead. The particle typically acts onlyto modify the density of the bead such that the material can be used fornon-packed bed applications. In some cases the solid particle providesrigidity and/or reduced gel volume.

Recently, materials have been developed with so-called “lids” or anouter layer of non-absorptive polysaccharide used to restrict theentrance to the porous structure, in order to avoid the binding of largemolecules while maintaining capacity for smaller species. Thesematerials have very low capacities for larger molecules as theirinternal surface area is not available to both species for binding.

Previous work has shown it possible to modify a symmetric polysaccharidestructure with a chemical modification in an asymmetric way. Thistechnique allows for the creation of a changing chemical environmentwithin the bead. Typically the modification is used to provide a neutrallayer on the outside of the bead. This prevents fouling of the outsideof the bead, especially in dirty feed streams such as those found inexpanded bed absorption (EBA). However, this technique does not changethe pore size of the bead, therefore resulting in a symmetric pore sizethroughout the bead.

Methods also have been developed to modify the pore size of porouschromatography media. Using these techniques, the creation of a bimodaldistribution of pore sizes is possible. However, this pore structure isevenly distributed throughout the bead and therefore does not createchemically unique regions within the bead to tune/alter observedselectivity.

Accordingly, a better media design is needed to improve biomoleculepurifications in which the separation is driven by more than onecharacteristic of the biomolecule.

SUMMARY OF THE INVENTION

The problems of the prior art have been overcome by the presentinvention, which provides an asymmetric chromatography media suitablefor separations applications, particularly as packed bed, fluidized bedor magnetized bed chromatography media. In certain embodiments, theasymmetric chromatography media comprises asymmetric particles,preferably beads, having at least two distinct layers, generally witheach having distinct controlled pore size distributions. Preferably oneof the distinct pore size distributions is in an internal region of theparticle, and the other is in an external region or coating on theparticle. These distinct pore size distributions can be modified withuniform or alternatively unique functional groups or mixtures offunctional groups. The present invention allows for the control overpore size distribution within an asymmetric porous particle by providinga distinct internal region, preferably in the form of a bead, and adistinct external region, preferably in the form of a coating on thebead.

The asymmetric beads in accordance with the present invention providerigidity and allow for control of the mass transfer path of packed-bedmedia. A rigid media can be stacked higher, thus attaining an overallhigher absorbing or exchanging capacity for the column. By shorteningthe diffusion path, the gel can be utilized at a much higher efficiencythan homogeneous beads. Under certain operating conditions, cored beadscan result in sharper elution peaks and less buffer consumption.

Another aspect of this invention is the ability to have a bead with twodistinct regions, each having one or more characteristics that isdifferent from the other. For example, it may simply be pore size thatdiffers between the first and second region. Alternatively and/oradditionally, it may be ligand type, density or mixture, media materialand/or percentage of agarose used in each region (when making an agarosebead) and the like that differs.

Another application of the cored beads of the present invention is influidized bed chromatography or magnetic chromatography where the coreprovides the required density (for fluidized beds) or the magneticproperties for the magnetized chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cross-sectional view of a symmetric agarose bead.

FIG. 1B is a cross-sectional view of an asymmetric agarose bead; and

FIG. 2A is a schematic view of an array of asymmetrical beads inaccordance with an embodiment of the present invention under low stress.

FIG. 2B is a schematic view of an array of asymmetrical beads inaccordance with an embodiment of the present invention under highstress.

DETAILED DESCRIPTION OF THE INVENTION

The asymmetric chromatographic media in accordance with the presentinvention enable the tuning of the mechanical and selectivity propertiesof the media to provide improved materials particularly suited forbiomolecule separations. In a preferred embodiment, the media includestwo distinct, controlled pore size distributions with unique chemicalmodification to each pore size region. One distinct pore sizedistribution is located in an internal region of media, and the other islocated at an external region such as a coating. Although the pore sizecan be the same in both regions, yielding a symmetric chromatographicmedia with regionally different chemical modification, preferably thepore sizes in the two regions are different. Most preferably, the poresize in the internal region is smaller than the pore size in theexternal region. The size of the pores is not particularly limited; forexample, the internal pore size can be small enough to effectively onlyadsorb small biomolecules (e.g., less than 10 KD molecular weight) orcan be open enough for large biomolecules. The internal and externalpore sizes can extend over a range necessary to affect an improvedchromatographic separation.

Suitable media materials include those typically used forchromatographic beads, including glass, and natural and syntheticpolymers such as styrene-divinyl benzene copolymer, agarose, agarosederivatives, agar, alginate, cellulose, cellulose derivatives, dextran,starch, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth,karaya gum, locust bean gum, xanthan gum, pectins, mucins, heparins, andgelatins. Agarose is particularly preferred. The beads can be formed ofthe same material throughout, or can be a composite of two or morematerials, preferably porous materials. For example, the bead cancomprise a glass or synthetic polymer bead or core on the inside and anagarose layer on the outside. Filler may be included to control density,for example. Where agarose is the media material in both the internaland external regions, the pore size distribution can be controlled byvarying the concentration of agarose. For example, a largerconcentration of agarose will result in a smaller pore size, and thusthe internal region can be 15% agarose while the external coating layercan be 6% agarose. Such an external layer will therefore adsorb largeproteins, such as IgG, while the internal core layer effectively (withinthe time scales of the chromatographic separation) excludes largeproteins.

More specifically, the cores of the particles of the present inventioncan be made of any material that is useful in chromatography. Forexample, the core may be a crossed linked agarose bead, a plastic,metal, glass or ceramic. Preferably when the finished bead is to havehigh rigidity, the core is selected from a material that is doesn't meltat the temperatures used in the manufacturing process and which isself-supportive. Suitable materials include but are not limited toplastics such as polystyrene, polyethylene, polypropylene, blends ofpolyethylene and polypropylene, multilayered polyethylene/polypropylenebeads, acrylics, polysulfones, polyethersulfones, PVDF or PTFE; glasssuch as borosilicate glass, alkali resistant glass and controlled poreglass, metals such as stainless steel, nickel, titanium, palladium andcobalt or various iron, iron containing or other magnetized metalsalloys and blends; and ceramics, such as silicate materials, zirconiaand various ceramic blends.

The cores are preferably of a generally spherical or irregularparticulate shape. Their diameter depends upon the size of bead onedesires but preferably is from about 30 microns to about 150 microns indiameter.

As is common in agarose bead manufacture, various additives can be usedto enhance production or add a property to the beads. One class ofadditives comprises volatile organics, miscible with the solution.Examples are monohydric alcohols such as methanol, ethanol, andpropanols. These can be used up to concentrations that give a slightlycloudy solution. Miscible ketones such as acetone can also be used, butcare must be used as the solubility of agarose is less in ketone-watermixtures. Any mixture of two or more of these materials is alsocontemplated.

A further class of additives comprises non-volatile miscible organics.Non-limiting examples of these included glycerine, ethylene glycol,methyl pentane diol, diethylene glycol, propylene glycol, triethyleneglycol, the methyl, ethyl, or n-butyl ethers of ethylene glycol, thedimethyl or diethyl ethers of ethylene glycol, ethylene glycol dimethylether acetate ethylene glycol diethyl ether acetate, diethylene glycolmethyl ether, diethylene glycol ethyl ether, diethylene glycol n-butylether, diethylene glycol dimethyl ether, diethylene glycol diethylether, diethylene glycol dimethyl ether acetate, diethylene glycoldiethyl ether acetate, N-methyl morpholine, N-ethyl morpholine, and thelike. Polyethylene glycols of low molecular weight are also examples ofmaterials that are in this class. Any mixture of two or more of thesematerials is also contemplated.

Another class of additives comprises water-soluble polymers, whichinclude by way of examples, polyvinyl pyrrolidone, polyvinyl alcohol,polyethylene glycols, dextrans, and water-soluble polyacylamides,including substituted polyacylamides, such as polydimethylacrylamideThese polymeric additives can be used as blends with the agarose in theinitial dissolution step, or they can be dissolved in the solution afterthe addition and dissolution of the agarose. Care must be taken not toadd an excessive amount of polymer, as coagulation of the solution mayoccur. Ratios of polymer to agarose of from about 0.1 to 10 arepossible. Preferred polymers are polyvinyl alcohol, dextrans andpolyacrylamides.

The media, particularly where comprised of agarose, may be crosslinkedif desired by any of the chemistries commonly used in the industry tocrosslink materials containing multiple hydroxyl groups, such aspolysaccharide beads, these chemistries being as non-limiting examples,epichlorohydrin or other multifunctional epoxy compounds, various bromylchemistries or other multifunctional halides; formaldehyde,gluteraldehyde and other multifunctional aldehydes, bis(2-hydroxyethyl)sulfone, dimethyldichloro-silane, dimethylolurea, dimethylolethylene urea, diisocyanates or polyisocyanates and the like.

It may also have one or more functionalities applied to it, includingligands, such as Protein A or Protein G, natural or recombinatorilyderived versions of either, modified versions of protein A or G torender them more caustic stable and the like, various chemical ligandssuch as 2-aminobenzimidazole (ABI), aminomethylbenzimidazole (AMBI),mercaptoethylpyridine (MEP) or mercaptobenzimidazole (MBI), or variouschemistries that render the agarose cationic, anionic, philic, phobic orcharged, as is well-known in the art of media formation.

Functional groups used in liquid chromatography that are adaptable tothe present invention include groups such as, but not limited to, ionexchange, bioaffinity, hydrophobic, groups useful for covalentchromatography, thiophilic interaction groups, chelate or chelating,groups having so called pi-pi interactions with target compounds,hydrogen bonding, hydrophilic, etc.

These groups may be added after the agarose bead has been formed andcrosslinked or they may be added to the initial solution and thecomposition of the initial solution is modified accordingly, such as pHbeing lowered or raised, so that the reaction to link the functionalgroups to the agarose occurs concurrently with the crosslinkingreaction.

These functionalities are commonly applied in two ways. First thesefunctionalities can be applied with the addition of anelectrophile-containing molecule which possesses the desired functionalgroup or a precursor thereof. Typically, the hydroxyl containing basematrix is activated with sodium hydroxide, which allows for efficientreaction of the base matrix with the aforementioned electrophile.Non-limiting examples include: bromopropane sulfonic acid, propanesultone, allyl glycidyl ether, allyl bromine, glycidyl trimethylammoniumchloride, butanediol diglycidyl ether, sodium chloroacetate.Alternatively, a nucleophilic group, such as an amine or thiol, can beadded to the base matrix using methods known in the art and then theabove electrophilic reagents can be used to modify the base matrix.Secondly, these functionalities can be applied with activation of thebase matrix with an electrophilic group including the followingnon-limiting samples, cyanogen bromide, activated carboxylic acids,aldehydes, esters, epoxides such as butanediol diglycidyl ether,epichlorohydrin, allyl bromide and allyl glycidyl ether, followed byreaction of the activated base matrix with the appropriate nucleophilicmolecule containing the functionality of choice. These nucleophiles canbe small molecules such as aminopropane sulfonic acid or larger entitiessuch as polyethyleneimine or proteins and peptides. In addition, all theabove chemistries can be added to the bead after hydrophilic spacer armsor “tentacles” such as dextran have been attached to the base matrix.This provides additional surface area for binding and further changesthe pore size and chemical environment within the bead's asymmetricstructure.

The asymmetric chromatography media of the present invention offerimproved mechanical properties over symmetric beads. Many commonchromatography media formed from polysaccharides are compressible. Thiscompressibility is initially determined by the porosity of the material.For example, commercial agarose beads are typically 4-6% agarose, whichmeans that the particles are 96-94% porous. Without chemicalmodification, these materials have very limited chromatographicapplications. Although after extensive crosslinking, these materials canbe rigid enough for many applications, the more porous the initialpolysaccharide bead, the more limited its mechanical properties will be.With the asymmetric media of the present invention, if the internal beadis much less porous than the external pore structure, the mechanicalproperties of the internal region is much greater than that of theexternal region. Under mechanical stress, the external region willcompress much like its symmetric counterparts, However, after theinitial compression, the internal regions of adjacent beads will beginto contact each other, as shown diagrammatically in FIG. 2. This “pointcontact” of the internal regions leads to a packed bed with themechanical compressibility of the internal bead structure. Because thisinternal structure can be less porous and more rigid, the net effect isa more porous or open external structure with the bed mechanicalcompressibility of a much more rigid material. If an incompressiblematerial is used as the internal pore structure, then the resultingchromatography bed will be incompressible after “point contact” of theinternal structures.

The selectivity of chromatography media for a given biomolecule ormixture thereof, is governed by a complex set of factors includingligand type, ligand density, media pore size, media hydrophobicity, etc.The selectivity of a symmetric bead could be viewed as “one dimensional”because the binding environment in the bead is uniform. Therefore, theselectivity of the media is driven by the ability of each individualbiomolecule to interact within the same binding environment. In otherwords, “molecule specific interactions” (species net charge,hydrophobicity, hydrogen bonding, etc.), not molecular size, drive theseparation of the different biomolecules. An advantage of an asymmetricchromatography bead is that the material contains at least two distinctchemical/binding environments in which species can bind. This structurecan be created by varying 1) the pore size between the internal andexternal pore structures, 2) the ligand type, ligand density and/orligand mixture, 3) media material (e.g. more philic outside/more phobicinside), and combinations of 1), 2) and 3). These materials provide “twodimensional” selectivity, because the separation is driven not only bythe molecule specific interactions, but also by the at least twodistinct regions within the media. For example, a weaker binding specieswill likely concentrate in a stronger binding environment or largerspecies could be excluded from the internal pore structure.

A specific example of an application of the novel media of the presentinvention is the separation of a Mab from host cell proteins (HCP).Mab's are higher molecular weight proteins (160 kD) and cannot easilydiffuse in 15% agarose gels. Accordingly, a 6% agarose bead with aninternal bead of 15% agarose provides an impurity “sink” in which HCPcan be adsorbed to one region (with a possibly unique chemicalmodification) while the Mab would be absorbed to the 6% structure. Thisprovides enhanced removal of common HCP impurity. In order to maximizethe binding capacity of the desired protein (Mab), the smaller pore sizeregion (the sink region) can be minimized to 20% of the total volume andthe more open pore region of the agarose bead can be modified with“tentacles” of a hydrophilic ligand carrier such as dextran. This candouble the capacity of the agarose bead for Mab.

Another advantage is the external region can be limited in thickness(1-15 microns) to allow for a shorter diffusional path during elution.This can provide a more narrow elution peak and possibly betterresolution.

A suitable process for making the media of the present inventioninvolves dissolving/melting the media material, such as agarose, in asuitable liquid, adding cores, preferably in bead form, so that thecores are coated with the agarose, mixing the coated cores with ahydrophobic liquid to form an emulsion and maintaining that emulsion ata temperature equal to or greater than the melting point of the agarose,passing it through a static mixer to create agarose droplets andsolidifying the agarose droplets in a second bath of hydrophobic liquid.The beads can then be washed and used or further processed to crosslinkthe agarose and/or add various functionalities onto the agarose as isknown in the art.

EXAMPLES Example 1A Asymmetric Agarose Bead with Unique ChemicalEnvironments and Uniform Pore Size

SP-Sepharose Fast Flow (6% crosslinked agarose) was coated with a 10-15micron layer of 6% agarose according to the following method: 50 ml ofthe beads were then mixed into 300 ml of 6% agarose solution (D-5Agarose from Hispanagar) to obtain a slurry. The agarose-beads mixturewas added to 1000 ml of mineral oil at 90° C. under constant agitationto obtain an emulsion in which the oil phase is continuous. The emulsionwas then pumped through a 0.5 inch (12.7 mm) diameter, 6 inches (152.4mm) long Kenics static mixer (KMR-SAN-12) at a flow rate of 3 L/min intomineral oil at 5° C. The resulting agarose beads had an estimatedexternal layer thickness of 10 um and the bead population waspredominantly single-cored (>50%). The beads were settled, washed withof water, ethanol and then water. The beads were crosslinked accordingto the method disclosed in Porath, Jerker and Janson, Jan-Christer andLaas, Torgny, Journal of Chromatography, 60 (1971) P. 167-177, thedisclosure of which is hereby incorporated by reference. The bead wasthen modified with bromoporane sulphonic acid (BPSA). In a jar, 10 gbeads, 30 ml of 5M NaOH, 7.2 g BPSA were added and agitated overnight at50° C. The beads were washed with 500 ml of Milli-Q quality water andthe BPSA process was repeated at second time. The beads were washed with500 ml of Milli-Q water and then stored in 20% ethanol.

Example 1B

SP-Sepharose Fast Flow (6% crosslinked agarose) was coated with a 10-15micron layer of 6% agarose as discussed in Example 1A. The beads werecrosslinked according to the method disclosed in Porath, Jerker andJanson, Jan-Christer and Laas, Torgny, Journal of Chromatography, 60(1971) P. 167-177. The bead was then modified with bromoporane sulphonicacid (BPSA). In a jar, 10 g beads, 30 ml of 5M NaOH, 7.2 g BPSA wereadded and agitated overnight at 50° C. The beads were washed with 500 mlof Milli-Q quality water and the BPSA process was repeated threeadditional times. The beads were washed with 500 ml of Milli-Q water andthen stored in 20% ethanol.

Example 1C.

SP-Sepharose Fast Flow (6% crosslinked agarose) was coated with a 10-15micron layer of 6% agarose as discussed in Example 1A. The beads werecrosslinked according to the method disclosed in Porath, Jerker andJanson, Jan-Christer and Laas, Torgny, Journal of Chromatography, 60(1971) P. 167-177. The bead was then modified with allyl glycidyl ether(AGE). In a jar, 10 g beads, 10 ml of 8M NaOH, 2 g AGE, 2 g Na₂SO₄ wereadded and agitated overnight at 25° C. The beads were washed with 500 mlof Milli-Q quality water and then stored in 20% ethanol.

Selectivity Testing

Two proteins of different net charge and molecular weight were used totest the nature of the selectivity or separation factor under typicalcation exchange (CEX) conditions. Pulses of a protein mixture containing0.5 mg/ml polyclonal IgG (Sigma) and 0.5 mg/ml lysozyme (Sigma) in 50 mMacetate buffer with NaCl to give conductivity 10 mS at pH 4.5 wereapplied to each sample column at 200 cm/hr (7 cm bed height, 0.66 cmdiameter). The pulse was then eluted at 200 cm/hr using a 20 CV NaClgradient starting at 10 mS and ending at 80 mS. The peak for eachprotein was recorded in terms of column volumes (CV) from the start ofthe elution gradient (dead volume was corrected). The data for the peakseparation are shown in Table 1:

TABLE 1 IgG PEAK LYSOZYME PEAK SEPA- SAMPLE (CV) PEAK (CV) RATION (CV)Example 1A 9.36 13.46 4.1 Example 1B 9.63 14.03 4.4 Example 1C 9.9513.65 3.7 SP-Sepharose 9.95 13.85 3.9 Fast Flow

The Table reports the peak positions in the elution gradient for bothIgG and lysozyme. The longer the delay in elution (greater CV), the morestrongly bound a species is to the media. The media were designed tohave differing chemical environments, mainly the density of sulfopropylligands. The difference in ligand density between the outside agaroseregion and the internal region is increasing in the following order:Example 1C<Example 1B<Example 1A. There is very little differencebetween the inside ligand density and outside ligand density in Example1C, each having essentially identical chemistry. The IgG peak positionfollows this general trend. The IgG peak elution occurs first for thebead with the lowest ligand density in the outer layer, while forExample 1C the elution occurs identically to the internal bead structurealone (SP-Sepharose control). Interestingly, the lysozyme and peakseparation do not show the same simple trend. However, it is clear thatthe peak separation is greater for the asymmetric beads with uniquechemical regions within the bead structure.

Example 2

Agarose beads (ABT Technologies) containing 10% agarose were crosslinkedaccording to Porath, et al., above. The bead was then modified withbromopropane sulphonic acid (BPSA). In a jar, 100 g beads, 300 ml of 5MNaOH, 72 g BPSA were added and agitated overnight at 50° C. The beadswere washed with 1500 ml of Milli-Q quality water and the BPSA processwas repeated two more times. The beads were washed with 1500 ml ofMilli-Q water and then stored in 20% ethanol. The beads were then coatedwith a layer of 6% agarose to form asymmetric agarose beads according tothe following method: 100 ml of the beads were then mixed into 600 ml of6% agarose solution (D-5 Agarose from Hispanagar) to obtain a slurry.The agarose-beads mixture was added to 2000 ml of mineral oil at 90° C.under constant agitation to obtain an emulsion in which the oil phase iscontinuous. The emulsion was then pumped through a 0.5 inch (12.7 mm)diameter, 6 inches (152.4 mm) long Kenics static mixer (KMR-SAN-12) at aflow rate of 3 L/min into mineral oil at 5° C. The resulting agarosebeads had an estimated external layer thickness of 10 um and the beadpopulation was predominantly single-cored (>50%). The beads weresettled, washed with of water, ethanol and then water. These beads werecrosslinked according to Porath, et al., above. The beads were thentreated with glycidyl trimethylammonium chloride (GTMAC). In a jar, 100ml of beads were added to 100 ml of 75% GTMAC. After mixing, 3.3 ml of50% NaOH was added and the reaction was shaken overnight at 25° C. Thebeads were washed with 1500 ml of Milli-Q water. The beads were thencoated with again with another layer of 6% agarose. The beads were thenshown a mixture of two dyes: methylene blue (1 mg/ml) and ponceau S (1mg.ml), in Milli-Q water. A sample of beads was shown only methyleneblue as a control. The beads were washed with Milli-Q water three times(100 ml). The dye location was recorded using a digital camera adaptedto a microscope lens.

The BPSA (negative charge) center 10% agarose region bound methyleneblue dye. The second region is positively charged (with GTMAC) and didnot bind methylene blue. However, upon exposure of the bead to a mixtureof methylene blue and the negatively charged dye ponceau S (red color),a blue center region was observed with a strong band of red where theGTMAC region is. The neutral outermost region contained some residualred dye from incomplete washing, but was less intense red as expected.

Example 3 Example 3A Asymmetric Agarose Bead with Unique ChemicalEnvironments and Two Distinct Pore Size Regions: Preparation of InternalStructure

A 15% agarose bead was made using the following method: 1000 ml of 15%agarose solution (D-5 Agarose from Hispanagar) was added to 2000 ml ofmineral oil containing 120 ml of Span 80 emulsifier in a first oil bathat 80° C. under constant agitation to obtain an emulsion in which theoil phase is continuous. The emulsion was then pumped through a 0.5 inch(12.7 mm) diameter, 6 inches (152.4 mm) long Kenics static mixer(KMR-SAN-12) at a flow rate of 3 L/min into a second bath of mineral oilat 5° C. Spherical homogeneous agarose beads were obtained with alargest particle diameter of 200 um. The beads were settled, washed withof water, ethanol and then water and sieved to yield a bead size rangeof 75-125 □m. The beads were then crosslinked according to the methoddisclosed in Porath, Jerker and Janson, Jan-Christer and Laas, Torgny,Journal of Chromatography, 60 (1971) P. 167-177, the disclosure of whichis hereby incorporated by reference. The beads were then modified withallyl glycidyl ether (AGE). In a jar, 120 g beads, 150 ml of 8M NaOH, 30g sodium sulfate and 100 g of AGE were added and agitated overnight at45° C. The beads were washed with 3×500 ml of Milli-Q quality water.

Example 3B Asymmetric Agarose Bead with Unique Chemical Environments andTwo Distinct Pore Size Regions: Preparation of Internal Structure

A portion of the beads made in Example 3A were modified to create acation exchange material. The beads were modified with sodiummeta-bisulfite. In a jar, 60 g beads, 47 ml of Milli-Q water, 7.9 g of50% wt NaOH and 23.4 g sodium meta-bisulfite were added and agitatedovernight at room temperature. The beads were washed with 3×500 ml ofMilli-Q quality water.

Example 3C Asymmetric Agarose Bead with Unique Chemical Environments andTwo Distinct Pore Size Regions: Preparation of External Structure

The beads from Example 3B were coated with 6% agarose according to thefollowing method: 50 ml of the beads were then mixed into 300 ml of 6%agarose solution (D-5 Agarose from Hispanagar) to obtain a slurry. Theagarose-beads mixture was added to 1000 ml of mineral oil at 90° C.under constant agitation to obtain an emulsion in which the oil phase iscontinuous. The emulsion was then pumped through a 0.5 inch (12.7 mm)diameter, 6 inches (152.4 mm) long Kenics static mixer (KMR-SAN-12) at aflow rate of 3 L/min into mineral oil at 5° C. The resulting agarosebeads had an estimated external layer thickness of 10 um and the beadpopulation was predominantly single-cored.(>50%). The beads weresettled, washed with of water, ethanol and then water and sieved toyield a bead size range of 75-1250 m. The beads were crosslinkedaccording to the method disclosed in Porath, Jerker and Janson,Jan-Christer and Laas, Torgny, Journal of Chromatography, 60 (1971) P.167-177, the disclosure of which is hereby incorporated by reference.The beads were washed with 3×500 ml of Milli-Q quality water.

Example 3D Asymmetric Agarose Bead with Unique Chemical Environments andTwo Distinct Pore Size Regions: Addition of Cation Exchange Ligands toExternal Structure

A portion of the beads from Example 3C were then modified withbromopropane sulfonic acid (BPSA). In a jar, 10 g beads, 30 ml of 5MNaOH, 7.2 g BPSA were added and agitated overnight at 50° C. The beadswere washed with 500 ml of Milli-Q quality water and then stored in 20%ethanol.

Example 3E Asymmetric Agarose Bead with Unique Chemical Environments andTwo Distinct Pore Size Regions: Addition of Cation Exchange Ligands toExternal Structure

A portion of the beads from Example 3C were then modified withbromopropane sulfonic acid (BPSA). In a jar, 10 g beads, 30 ml of 5MNaOH, 7.2 g BPSA were added and agitated overnight at 50° C. The beadswere washed with 500 ml of Milli-Q quality water and the BPSA processwas repeated one additional time. The beads were washed with 500 ml ofMilli-Q water and then stored in 20% ethanol.

Example 3F Asymmetric Agarose Bead with Unique Chemical Environments andTwo Distinct Pore Size Regions: Addition of Cation Exchange Ligands toExternal Structure

A portion of the beads from Example 3C were then modified withbromopropane sulfonic acid (BPSA). In a jar, 10 g beads, 30 ml of 5MNaOH, 7.2 g BPSA were added and agitated overnight at 50° C. The beadswere washed with 500 ml of Milli-Q quality water and the BPSA processwas repeated two additional times. The beads were washed with 500 ml ofMilli-Q water and then stored in 20% ethanol.

Selectivity Testing.

Two proteins of different net charge and molecular weight were used totest the nature of the selectivity or separation factor under typicalcation exchange (CEX) conditions. A mixture of a protein containing 0.5mg/ml polyclonal IgG (Sigma) and 0.5 mg/ml lysozyme (Sigma) in 50 mMacetate buffer with NaCl to give conductivity 10 mS at pH 4.5 wereapplied to each sample column at 200 cm/hr (7 cm bed height, 0.66 cmdiameter) such that the net protein loaded on the media was 10 mg/mL.The protein mixture was then eluted at 200 cm/hr using a 30 CV NaClgradient starting at 10 mS and ending at 80 mS. The peak for eachprotein was recorded in terms of column volumes (CV) from the start ofthe elution gradient (dead volume was corrected). The data for the peakseparation are shown in Table 2:

TABLE 2 IgG PEAK LYSOZYME PEAK SEPA- SAMPLE (CV) PEAK (CV) RATION (CV)Example 3D 19.7 25.7 6.6 Example 3E 18.6 26.7 8.1 Example 3F 18.7 26.88.1 SP-Sepharose 20.3 26.3 6 Fast Flow

Table 2 reports the peak positions in the elution gradient for both IgGand lysozyme. The longer the delay in elution (greater CV), the morestrongly bound a species is to the media. The media were designed tohave differing chemical and physical environments, mainly the density ofsulfopropyl ligands and internal structure pore size. For Example 3D,the selectivity is similar to that of a standard commercial resin(Sepharose Fast Flow). However, Examples 3E and 3F show an increasedseparation of the two protein peaks as a result of the asymmetric beadstructure with unique chemical and pore size regions.

Example 5 Agarose Cored Bead

75 ml of 15% cross-linked agarose beads of 100 um average diameter (Madeusing process patent to be file on same day) was mixed with 100 ml of 4%agarose solution (Hispanagar D5) to obtain a slurry. The agarose-beadmixture was added to 2000 ml of mineral oil at 80° C. under constantagitation to obtain an emulsion in which the oil phase is continuous.The emulsion was then pumped through a 0.5″ diameter, 6″ long Ross ISGstatic mixer at a flow rate of 3 L/min into mineral oil at 5° C. Theresulting asymmetric beads had an estimated external region thickness of10 um and the bead population was predominantly single-cored. (>60%)

What is claimed is:
 1. A method of separating a monoclonal antibody froma host cell protein, the method comprising contacting a mixture ofantibody and host cell protein with an asymmetric chromatographyparticle comprising an internal region and an external region, each ofthe internal region and the external region has a different percentagarose, wherein the monoclonal antibody is absorbed onto the externalregion and the host cell protein is adsorbed onto the internal region.2. The method of claim 1, wherein the external region comprises anagarose concentration of 6% and the internal region comprises an agaroseconcentration of 15%.
 3. The method of claim 1, wherein the in internalregion higher percentage of agarose than the external region.
 4. Themethod of claim 1, wherein the external region comprises a thickness of1 to 15 microns.
 5. The method of claim 1, wherein the chromatographyparticle is modified with a hydrophilic ligand carrier.
 6. The method ofclaim 1, wherein the chromatography particle is modified with ahydrophilic ligand carrier and the hydrophilic ligand carrier isdextran.
 7. The method of claim 1 wherein the agarose of the internaland external region is crosslinked.
 8. The method of claim 1 wherein theagarose of the internal and external region is crosslinked and whereinthe chromatography particle is modified with a hydrophilic ligandcarrier and the hydrophilic ligand carrier is dextran.